Isolation Of The Gene Of Interest By

1. For cultured cells, aspirate culture medium and rinse the cells twice in PBS. Add 10 ml/60 mm culture plate (with approximately 105 cells) of DNA isolation buffer to the cells and allow cells to be lysed for 5 min at room temperature with shaking at 60 rpm. For tissues, grind 1 g tissue in liquid N2 to fine powder and transfer the powder into a tube containing 15 ml of DNA isolation buffer. Allow cells to be lysed for 10 min at room temperature with shaking at 60 rpm.

2. Incubate at 37°C for 5 h or overnight to degrade proteins, and extract genomic DNA.

3. Allow the lysate to cool to room temperature and add three volumes of 100% ethanol precooled at -20°C to the lysate. Gently mix it and allow DNA to precipitate at room temperature with slow shaking at 60 rpm. Precipitate DNA in appearance of white fibers should be visible in 20 min.

4. "Fish" out the DNA using a sterile glass hook or spin down the DNA. Rinse the DNA twice in 70% ethanol and partially dry the DNA for 40 to 60 min at room temperature to evaporate ethanol. The DNA can be directly subjected to restriction enzyme digestion without being dissolved in TE buffer or dd.H2O. We usually overlay the precipitated DNA with an appropriate volume of restriction enzyme digestion cocktail and incubate the mixture at the appropriate temperature for 12 to 24 h. The digested DNA is mixed with an appropriate volume of DNA loading buffer and is ready for agarose gel electrophoresis.

Reagents Needed

DNA Isolation Buffer

100 mM NaCl

1 mg/ml Protease K (freshly added)

0.1% (w/v) N-Lauroylsarcosine

Partial Digestion of Genomic DNA Using Sau3A I

It is necessary to partially cut the high molecular weight of genomic DNA with a four-base cutter, Sau3A I, to increase the efficiency of PCR.

Optimization of Partial Digestion of Genomic DNA with Sau3A I

In order to determine the amount of enzyme used to digest the high molecular weight

DNA into 20- to 30-Kb fragments, small-scale reactions, or pilot experiments, should be carried out.

3. Carry out 10 individual, small-scale digestion reactions on ice in the order listed below:

Components Tube Number

1

2

3

4

5

6

7

8

9

10

Genomic DNA

1(mi)

1

1

1

1

1

1

1

1

1

(1 mg/ml)

10X Sau3A I buffer

5(mi)

5

5

5

5

5

5

5

5

5

dd.H2O (ml)

39

39

39

39

39

39

39

39

39

39

Sau3A I dilution

5(mi)

5

5

5

5

5

5

5

5

5

in the same order

as in (b) above

Final volume (ml)

50

50

50

50

50

50

50

50

50

50

Note: The final Sau3A I concentration used in tube 1 to tube 10 should be 1, 0.1, 0.05, 0.025, 0.015, 0.0125, 0.01, 0.0085, 0.005, and 0.0035 unit/mg DNA, respectively.

Note: The final Sau3A I concentration used in tube 1 to tube 10 should be 1, 0.1, 0.05, 0.025, 0.015, 0.0125, 0.01, 0.0085, 0.005, and 0.0035 unit/mg DNA, respectively.

4. Incubate the reactions at the same time at 37°C for 30 min. Place the tubes on ice and add 2 ml of 0.2 M EDTA buffer (pH 8.0) to each tube to stop the reaction.

5. While the reactions are performed, prepare a large size of 0.4% agarose gel in 1X TBE buffer.

6. Add 10 ml of 5X DNA loading buffer to each of the 10 tubes containing the digested DNA prepared in Step (d).

7. Carefully load 30 ml of each sample to the wells in the order of 1 to 10. Load DNA markers (e.g., 1DNA Hind III markers) to the left or the right well of the gel to estimate the sizes of digested DNA.

8. Carry out electrophoresis of the gel at 2 to 5 V/cm until the bromphenol blue reaches the bottom of the gel.

9. Photograph the gel under UV light and find the well that shows the maximum intensity of fluorescence in the desired DNA size range of 20 to 30 Kb.

Large-Scale Preparation of Partially Digested Genomic DNA

1. Based on the optimal conditions established above, carry out a large-scale digestion of 50 mg of high-molecular-weight genomic DNA using half units of Sau3A I/mg DNA that produced the maximum intensity of fluorescence in the DNA size range of 20 to 30 Kb. The DNA concentration, time and temperature should be exactly the same as those used for the small-scale digestion. For instance, if tube 7 (0.01 unit of Sau3A I per microgram DNA) in the small-scale digestion of the DNA shows a maximum intensity of fluorescence in the size range of 20 to 30 Kb, the large-scale digestion of the same DNA can be carried out as follows. Genomic DNA (1mg/ml), 50 ml

10X Sau3A I buffer, 250 ml dd.H2O, 1.95 ml

Diluted Sau3A 1, 250 ml

Final volume of 2.5 ml

2. Incubate the reactions at 37°C in a water bath for 30 min. Stop the reaction by adding 20 ml of 0.2M EDTA buffer (pH 8.0) and place the tube on ice until use.

3. Add 2 to 2.5 volumes of chilled 100% ethanol to precipitate the DNA at -70°C for 30 min.

4. Centrifuge at 12,000 x g for 10 min at room temperature. Carefully decant the supernatant and briefly rinse the DNA pellet with 5 ml of 70% ethanol. Dry the pellet under vacuum for 8 min. Dissolve the DNA in 50 ml of TE buffer. Take 5 ml of the sample to measure the concentration at A260 nm and store the DNA sample at -20°C prior to use.

A pair of forward and reverse primers can be designed based on the 5'-UTR or 3'-UTR sequences of a known cDNA. The forward primer can be designed in the 5'-UTR region in the 5' ^ 3' direction with 20 to 30 bases, which should be complementary to the first or (-)strand of cDNA template. The reverse primer is designed in the 3'-UTR region in the 5' ^ 3' direction with 20 to 30 bases, which should be complementary to the second or (+)strand of cDNA template (Figure 2.6A). Alternatively, forward and reverse primers can be designed based on the very N-terminal and C-terminal amino acid sequences if the cDNA sequence is not known (Figure 2.6B).

Amplification and Isolation of Promoter Sequence

To obtain the promoter sequence of interest, a reverse primer can be designed according to the 5' end sequence of a known cDNA or based on the N-terminal amino acid sequence. This primer will be employed to anneal with a DNA strand of the denatured genomic DNA fragments. The primer facilitates the synthesis of a new strand, which is complementary to the template and includes the promoter

FP

-►

El

E2

E3

I l

I 2

I l

I 2

FIGURE 2.6 Design of primers for amplification and isolation of exon and intron sequences. (A) Forward primer (FP) and reverse primer (RP) are designed from the 5'-UTR and 3'-UTR sequences of a known cDNA. (B) FP and RP are designed based on the very N-terminal or C-terminal amino acid sequence. E: exon; I: intron.

3' DNA fragment

Promoter region ^

Reverse primer Annealing of specific primer and PCR 3'--- 5'

Promoter region j Addition of oligo(dC) to 3'-end by

3' (dC)n Promoter region |5 ' terminal transferase

Promoter region

(dG)n anchor primer i

Annealing of forward and reverse primers, PCR

Promoter region

Anchor or fowari pnmir ^Amplification by PCR Promoter region

Promoter region

Anchor or fowari pnmir ^Amplification by PCR Promoter region

Subcloning of PCR products

FIGURE 2.7 Diagram of amplification and isolation of promoter region of interest by PCR.

Subcloning of PCR products

Characterization of PCR products

FIGURE 2.7 Diagram of amplification and isolation of promoter region of interest by PCR.

sequence. Following addition of oligo(dC) to the 3' end of the new strand, an oligo(dG) anchor primer or forward primer in the cloning site of the vector will facilitate the synthesis of the DNA strand complementary to the new strand. As a result, major PCR products including the promoter sequence will be obtained (Figure 2.7).

Amplification of Specific DNA Fragments by PCR

1. Prepare a PCR cocktail in a 0.5-ml microcentrifuge tube.

Forward primer, 5 to 8 pmol (30 to 40 ng) depending on the size of primer

(20- to 26-mer, 10 to 30 ng/ml) Reverse primer, 5 to 8 pmol (30 to 40 ng) depending on the size of primer

2. Carefully overlay the mixture with 30 ml of mineral oil to prevent evaporation of the samples during the PCR amplification. Place the tubes in a thermal cycler and perform PCR.

Cycling (35 cycles)

Predenaturation Denaturation Annealing Extension Last

95°C, 3 min 95°C, 1 min 60°C, 1 min 72°C, 2 min 4°C

Purification of PCR Products by Agarose Gels

Carry out electrophoresis and elution of PCR products as described earlier in this chapter. Due to different sizes of genomic DNA fragments, multiple PCR bands may be shown in agarose gels. These bands should be individually isolated for characterization.

SUBCLONING OF CDNA OR GENE OF INTEREST Detailed protocols are described in Chapter 4.